Intracellular calcium concentration is known to play a significant role in both physiological and pathophysiological conditions. Recently, much attention has been given to the determination of free intracellular calcium concentration using flow cytometry. Newly developed fluorescent probes, such as Indo-1 which exhibits a large spectral emission shift when it chelates Ca++ ions have been instrumental in the development of this application. However, because calibration of the flow cytometer response with respect to the ratio of the FL1 (405 nm peak) and the FL2 (485 nm peak) cytometry has been limited. The present proposal focuses on the development of an Indo-1 Microbead Standards Kit consisting of Indo-1 coated microbeads suspended in a series of solutions containing pre-determined concentrations of free Ca++. Using this kit the ratio of the FL1 and FL2 emission fluorescence of Indo-1 microbeads can be directly correlated with free calcium concentration. Such a kit will not only make calibration of flow cytometers more convenient, but will also help to normalize data over time and among laboratories. A more accurate determination of cytosolic calcium concentration will undoubtedly improve our understanding of the biological relevance of alterations in intracellular calcium concentration.